High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Physiological aspects of surface-immobilized Catharanthus roseus cells

Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O₂ consumption and CO₂ production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures. Offprint requests to: Denis Rho     

Influence of different ammonium,lactate and glutamine concentrations on CCO cell growth

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.     

A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth

The rates of consumption of oxygen and glucose by EMT6/Ro cells in multicellular spheroids were measured at various times during normal growth. In situ spheroid cellular consumption rates were similar to those of exponentially growing single cells up to a spheroid diameter of 150 micron. Further growth resulted in decreases in the rates of both oxygen and glucose consumption which were correlated with the increase in spheroid diameter and cell number. At a diameter of 1300 micron, both rates of cellular consumption had decreased by a factor of 2.5. The rates of consumption per unit of nonnecrotic spheroid volume decreased in a similar manner. Measurements with single cells demonstrated that the rate of oxygen consumption was coupled with glucose concentration, and vice versa. The rates of consumption for cells dissociated from small spheroids indicated that there was some effect of the spheroid environment. As the spheroids grew, however, association in the spheroid structure accounted for a smaller proportion of the total observed reduction in the rates of nutrient consumption. The presence of central necrosis also appeared to have no effect on the rates of consumption of these nutrients. Spheroid-derived cells showed a decrease in cell volume with growth as the cells accumulated in a quiescent state. Measurements with single cells demonstrated that oxygen and glucose consumption were correlated with cell volume and with the development of nonproliferating cells. We conclude that the observed decrease in oxygen and glucose consumption with growth in spheroids is largely due to the progressive accumulation of cells in a quiescent state characterized by an inherently lower cellular rate of nutrient utilization.     

Hybridoma growth and antibody production as a function of cell density and specific growth rate in perfusion culture

Steady state metabolic parameters for hybridoma cell line H22 were determined over a wide range of cell densities and specific growth rates in a filtration based homogeneous perfusion reactor. Operating the reactor at perfusion rates of 0.75, 2.0, and 2.9 day(-1)(each at four different specific growth rates), viable cell densities as high as 2 x 10(7) cells/mL were obtained. For the cell line under investigation, the specific monoclonal antibody production rate was found to be a strong function of the viable cell density, increasing with increasing cell density. In contrast, most of the substrate consumption and product formation rates were strong functions of the specific growth rate. Substrate metabolism became more efficient at high cell densities and low specific growth rates. The Specific rates of metabolite formation and the apparent yields of lactate from glucose and ammonia from glutamine decreased at low specific growth rates and high cell densities. While the specific oxygen consumption rate was independent of the specific growth rate and cell density, ATP production was more oxidative at lower specific growth rate and higher cell density. These observed shifts are strong indications of the production potential of high-density perfusion culture. (c) 1995 John Wiley & Sons, Inc.     

Cellulase production by a solid state culture system

Production of cellulase using solid culture systems of Trichoderma reesei QM9414 and Sporotrichum cellulophilum on wheat bran was studied. By using moisture-controlled solid culture equipment, the effect of water content of wheat bran on cell growth and cellulase production was investigated. Cellular biomass grown on solid substrate was estimated by measuring oxygen consumption rate and glucosamine content of the cells. These parameters were shown to have a good linear correlation with the specific growth rate. This reliable method of estimating the cell growth rate enabled us to simulate the enzyme production in a solid culture system by means of multiple linear regression analysis which takes into account of the water content, cell mass, and the oxygen consumption rate as variables. The cell growth and cellulase production were maximized at different water content of the medium. A high water content, 57% for T. reesei and 70% for S. cellulophilum, favored mycelial growth, while the maximum cellulase activity was obtained at a lower water content such as 50% for both fungi. It was observed that cellulase production by T. reesei depended on the culture conditions that support the optimal growth rate for the maximum enzyme production.     

Changes in growth characteristics of mouse ascites tumor AISM during the process of passage in vivo

The growth characteristic of mouse ascites tumour AISM was observed on two distant passages (35th and 117th) in vivo. The following growth differences were established; the duration of life time of tumour bearing mice is less at the 35th passage (8 days) in comparison to the 117th passage (12 days); the common tumour cell mass at the terminal stage of life of tumour is more than 10 times less at 35th passage (10(8) cells) than at 117th passage (1.2.10(9) cells). The growth rate at 35th passage increases to the 4th day and at 117th passage to the 6th day. It is suggested that the tumour growth rate and the final size of tumor cell mass depend on the cell ploidity and chalone growth control.     

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O₂, CO₂, H₂O, and N₂ contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.     

人皮肤成纤维细胞在不同培养系统中的生长代谢特性

大面积烧伤病人及多种皮肤溃疡病人很难用自体皮肤移植来进行治疗.早期治疗方法采用尸体来源的皮肤移植,但由于来源有限、且有传播疾病的危险,因此应用组织工程技术构建生物活性人工皮肤已成为近十几年来在组织工程和创伤治疗领域的研究热点,目前已有几种人工皮肤成功地走向临床[1].然而,在构建大面积皮肤组织过程中,如何大量制备皮肤种子细胞仍然是一大棘手的难题,成为人体皮肤组织工程迫切需要解决的技术关键.获得大量扩增的皮肤细胞,解决种子细胞的供应问题,是构建人工皮肤的一个关键.     

Analysis of cell growth in a polymer scaffold using a moving boundary approach

Two mathematical models of chondrocyte generation and nutrient consumption are developed to analyze the behavior of cell growth in a biodegradable polymer matrix. Substrate reaction and diffusion are analyzed in two regions: one consisting of cells and nutrients and the other consisting of only nutrients. A pseudo-steady state approximation for the transport of nutrients in these two regions is utilized. The rate of growth is determined by a moving boundary equation that equates the rate at which the interfacial region between the cells and the void space moves to a substrate dependent growth reaction. The change in the location of this interfacial region with time therefore depicts the rate at which the cells propagate. The two limiting cases discussed in this article represent extremes in how the cells will grow in the polymer matrix; one case assumes that cells grow inward from the external boundary, and the other case assumes that cells grow parallel to the external boundary. The results of both models are compared to experimental data found in the literature. It is found through these comparisons that the model parameters, including the unit cell spacing parameter L, the metabolic rate constant k, the growth rate constant k(G), and external mass transfer coefficient, K, may vary as the thickness of the polymer matrix is changed, however, unrealistic and large changes in the diffusion coefficients were required to account for the full range of experimental data. Furthermore, these results suggest modification of the functional form of the growth kinetics to include substrate or product inhibition, or death terms. Based upon diffusion/reaction concepts, these models for cell growth in a biodegradable polymer give bounds for the upper and lower limits of the cellular growth rate and nutrient consumption in a polymer matrix and will aid in the development of more extensive models. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 422-432, 1997.     

Analysis of initiation of sites of cell wall growth in Streptococcus faecium during a nutritional shift

Three-dimensional reconstruction methods were applied to electron micrographs of Streptococcus faecium to study the initiation of cell wall growth sites during a nutritional shift experiment. Upon lowering the mass doubling time from 76 to 33 min by the addition of excess glutamate, the formation of new cell wall growth sites accelerated above the old steady-state rate at about the same time (10 to 15 min) as did mass, RNA, protein, cell numbers, and autolytic capacity but considerably before DNA (30 min) and peptidoglycan (20 min) synthesis did. During the shift, the average range of cell volumes over which new wall growth sites were introduced did not change significantly. However, upon the shift there was an increase in the frequency of cells having new sites, which was due to the faster-growing cells initiating more new sites in peripheral locations before division. After a transition period, the number of new sites per milliliter of culture increased at a rate that paralleled that of the culture mass. These findings support a model in which new sites are introduced when cells grow to a relatively constant, growth rate-independent size, while the rate at which sites form and grow increases with the growth rate. In this model, chromosome synthesis does not regulate the formation of new sites of cell wall growth, but existing sites cannot be completed until rounds of chromosome synthesis are completed.     

In situ oxygen consumption rates of cells in V-79 multicellular spheroids during growth

The rate of consumption of oxygen by V-79 cells in multicellular spheroids was measured as a function of the spheroid diameter. In situ consumption was equal to that of exponentially growing cells for spheroids less than 200 micron in diameter. The rate of oxygen consumption decreased for cells in spheroids between 200 and 400 micron diameter to a value one-fourth the initial, then remained constant with further spheroid growth. Comparison of consumption rates for spheroid-derived cells before and after dissociation from the spheroid structure indicated that the spheroid microenvironment accounted for only 20% of the change in oxygen consumption rate. Cell-cell contact, cell packing, and cell volume were not critical parameters. Plateau-phase cells had a fivefold lower rate of oxygen consumption than exponential cells, and it is postulated that the spheroid quiescent cell population accounts for a large part of the intrinsic alteration in oxygen consumption of cells in spheroids. Some other mechanism must be involved in the regulation of cellular oxygen consumption in V-79 spheroids to account for the remainder of the reduction observed in this system.     

Increase in cell mass during the division cycle of Escherichia coli B/rA.

Increase in the mean cell mass of undivided cells was determined during the division cycle of Escherichia coli B/rA. Cell buoyant densities during the division cycle were determined after cells from an exponentially growing culture were separated by size. The buoyant densities of these cells were essentially independent of cell age, with a mean value of 1.094 g ml-1. Mean cell volume and buoyant density were also determined during synchronous growth in two different media, which provided doubling times of 40 and 25 min. Cell volume and mass increased linearly at both growth rates, as buoyant density did not vary significantly. The results are consistent with only one of the three major models of cell growth, linear growth, which specifies that the rate of increase in cell mass is constant throughout the division cycle.     

Variations in tumor cell growth rates and metabolism with oxygen concentration, glucose concentration, and extracellular pH.

Tumors and multicellular tumor spheroids can develop gradients in oxygen concentration, glucose concentration, and extracellular pH as they grow. In order to calculate these gradients and assess their impact on tumor growth, it is necessary to quantify the effect of these variables on tumor cell metabolism and growth. In this work, the oxygen consumption rates, glucose consumption rates, and growth rates of EMT6/Ro mouse mammary tumor cells were measured at a variety of oxygen concentrations, glucose concentrations, and extracellular pH levels. At an extracellular pH of 7.25, the oxygen consumption rate of EMT6/Ro cells increased by nearly a factor of 2 as the glucose concentration was decreased from 5.5 mM to 0.4 mM. This effect of glucose concentration on oxygen consumption rate, however, was slight at an extracellular pH of 6.95 and disappeared completely at an extracellular pH of 6.60. The glucose consumption rate of EMT6/Ro cells increased by roughly 40% when the oxygen concentration was reduced from 0.21 mM to 0.023 mM and decreased by roughly 60% when the extracellular pH was decreased from 7.25 to 6.95. The growth rate of EMT6/Ro cells decreased with decreasing oxygen concentration and extracellular pH; however, severe conditions were required to stop cell growth (0.0082 mM oxygen and an extracellular pH of 6.60). Empirical correlations were developed from these data to express EMT6/Ro cell growth rates, oxygen consumption rates, and glucose consumption rates, as functions of oxygen concentration, glucose concentration, and extracellular pH. These empirical correlations make it possible to mathematically model the gradients in oxygen concentration, glucose concentration, and extracellular pH in EMT6/Ro multicellular spheroids by solution of the diffusion/reaction equations. Computations such as these, along with oxygen and pH microelectrode measurements in EMT6/Ro multicellular spheroids, indicated that nutrient concentration and pH levels in the inner regions of spheroids were low enough to cause significant changes in nutrient consumption rates and cell growth rates. However, pH and oxygen concentrations measured or calculated in EMT6/Ro spheroids where quiescent cells have been observed were not low enough to cause the cessation of cell growth, indicating that the observed quiescence must have been due to factors other than acidic pH, oxygen depletion, or glucose depletion.     

Increasing batch-to-batch reproducibility of CHO-cell cultures using a model predictive control approach

By means of a model predictive control strategy it was possible to ensure a high batch-to-batch reproducibility in animal cell (CHO-cell) suspensions cultured for a recombinant therapeutic protein (EPO) production. The general control objective was derived by identifying an optimal specific growth rate taking productivity, protein quality and process controllability into account. This goal was approached indirectly by controlling the oxygen mass consumed by the cells which is related to specific biomass growth rate and cell concentration profile by manipulating the glutamine feed rate. Process knowledge represented by a classical model was incorporated into the model predictive control algorithm. The controller was employed in several cultivation experiments. During these cultivations, the model parameters were adapted after each sampling event to cope with changes in the process’ dynamics. The ability to predict the state variables, particularly for the oxygen consumption, led to only moderate changes in the desired optimal operational trajectories. Hence, nearly identical oxygen consumption profiles, cell and protein titers as well as sialylation patterns were obtained for all cultivation runs.     

The initiation mass for DNA replication in Escherichia coli K-12 is dependent on growth rate.

It is widely accepted that the initiation mass of Escherichia coli is constant and independent of growth rate, and therefore is an important parameter in the regulation of initiation of DNA replication. We have used flow cytometry to measure the initiation mass of E. coli K-12 cells as a function of growth rate. The average initiation mass was determined by two methods: (i) from a mathematical relationship between average cell mass, cell age at initiation and number of origins present in the cells, and (ii) directly from the cell mass distribution. The light scattering signal from individual cells and the protein content per cell were employed as measures of cell mass. The initiation mass was found to increase monotonically with decreasing growth rate, being 1.6 times higher (light scattering) or 2.1 times higher (protein content) at 0.3 than at 2.5 doublings per hour. We conclude that the initiation mass is dependent on growth rate. This finding indicates that the control for timing of initiation is not governed by a direct connection between mass accumulation and the molecule(s) determining initiation of replication.     

Relationship of hepatocyte ploidy levels with body size and growth rate in mammals.

To elucidate possible causes of the elevation of genome number in somatic cells, hepatocyte ploidy levels were measured cytofluorimetrically and related to the organismal parameters (body size, postnatal growth rate, and postnatal development type) in 53 mammalian species. Metabolic scope (ratio of maximal metabolic rate to basal metabolic rate) was also included in 23 species. Body masses ranged 10(5) times, and growth rate more than 30 times. Postnatal growth rate was found to have the strongest effect on the hepatocyte ploidy. At a fixed body mass the growth rate closely correlates (partial correlation analysis) with the cell ploidy level (r = 0.85, P < 10(-6)), whereas at a fixed growth rate body mass correlates poorly with ploidy level (r = -0.38, P < 0.01). The mature young (precocial mammals) of the species have, on average, a higher cell ploidy level than the immature-born (altricial) animals. However, the relationship between precocity of young and cell ploidy levels disappears when the influences of growth rate and body mass are removed. Interspecies variability of the hepatocyte ploidy levels may be explained by different levels of competition between the processes of proliferation and differentiation in cells. In turn, the animal differences in the levels of this competition are due to differences in growth rate. A high negative correlation between the hepatocyte ploidy level and the metabolic scope indicates a low safety margin of organs with a high number of polyploid cells. This fact allows us to challenge a common opinion that increasing ploidy enhances the functional capability of cells or is necessary for cell differentiation. Somatic polyploidy can be considered a "cheap" solution of growth problems that appear when an organ is working at the limit of its capabilities.     

Growth of immobilised plant cells in reticulate polyurethane foam matrices

Summary An inoculum of initially freely suspended cell aggregates ofCapsicum frutescens was immobilised in porous polyurethane foam matrices. Subsequent growth and substrate consumption of these immobilised cells in batch culture were measured and compared with those of suspension cultures. The results showed that the maximum specific growth rate of freely suspended cells was slightly higher than that of immobilised cells but the overall growth patterns and final cell yields were similar.     

Optimization of the cell seeding density and modeling of cell growth and metabolism using the modified Gompertz model for microencapsulated animal cell culture

Cell microencapsulation is one of the promising strategies for the in vitro production of proteins or in vivo delivery of therapeutic products. In order to design and fabricate the optimized microencapsulated cell system, the Gompertz model was applied and modified to describe the growth and metabolism of microencapsulated cell, including substrate consumption and product formation. The Gompertz model successfully described the cell growth kinetics and the modified Gompertz models fitted the substrate consumption and product formation well. It was demonstrated that the optimal initial cell seeding density was about 4-5 x 10(6) cells/mL of microcapsule, in terms of the maximum specific growth rate, the glucose consumption potential and the product formation potential calculated by the Gompertz and modified Gompertz models. Modeling of cell growth and metabolism in microcapsules provides a guideline for optimizing the culture of microencapsulated cells.     

Unbalanced cell growth and increased protein synthesis induced by chemotherapeutic agents

Unbalanced cell growth as manifested by an increase in cellular volume and in cellular dry mass following exposure to a variety of chemotherapeutic agents has been shown for neoplastic cells in vitro and human leukemic cells in vivo. The purpose of the present investigation was to test the hypothesis that unbalanced cell growth results from a disassociation of cell growth and cell division due to the blocking effect of chemotherapeutic agents. Monolayer cultures of CHO fibroblasts were studied in terms of their response to two chemotherapeutic agents that differ significantly in their mode of action, adriamycin and chlorambucil. Following exposure to these drugs, cell volume increased at a rate of from 1% to 4% per h; the total cell protein increased at a rate of from 4% to 7% per h. These changes were observed in both log and stationary phase cultures. Thus exposure to adriamycin and chlorambucil was followed by a more rapid rate of protein synthesis relative to the rate of degradation, resulting in larger cells with more protein whether or not the cells were actively in the division cycle. This is inconsistent with the hypothesis that unbalanced growth results simply from a disassociation of the cell division cycle from cell growth. These observations suggest that a final common pathway in the mode of action of chemotherapeutic agents may be the induction of unscheduled protein synthesis resulting in unbalanced cell growth.